[Objective] This study aimed to scientifically improve the 1-deoxynojimycin (DNJ) yield of mulberry leaves in spring. [Method] “3414” experimental design was adopted to investigate the effects of different fertilizer application rates of nitrogen, phosphorus and potassium on the DNJ content in mulberry leaves. The regression relationship was further simulated with DNJ yield as the objective function and fertil- izer application rates of nitrogen, phosphorus and potassium as the regulatory factors. [Result] The results showed that nitrogen, phosphorus and potassium all have great effects on the DNJ content in mulberry leaves at different parts of mulberry tree (Morus alba); the DNJ content in mulberry leaves increases with the increasing single-factor fertilizer application rate and reaches the maximum under N2P2K2 level; different fertilizer application rates and combinations of nitrogen, phosphorus and potassium can significantly affect the DNJ content in mulberry leaves; according to the simulated fertilizer effect function, the optimal economic yield of total DNJ in mul- berry leaves was 23.23 kg/hm^2, the recommended fertilization indicators were 286.11 kg/hm^2 N, 95.22 kg/hm^2 P2O5 and 159.62 kg/hm^2 K20. [Conclusion] After confirmation in productive practice, this optimal fertilization program can provide reference for the construction of mulberry plantation with high yield and high quality of DNJ.